The present invention relates to methods for detecting endotoxins in a sample.
Endotoxin (ET) describes a family of lipopolysaccharides which together with proteins and phospholipids form the outer cell wall of Gram-negative bacteria. Endotoxins occur exclusively in this bacterial group and play an important role in the organisation, stability and barrier function of the outer membrane. Numerous bacteriophages use endotoxin or general lipopolysaccharide for specific detection of their host bacteria.
All endotoxin variants comprise a heteropolysaccharide which is bonded covalently to lipid A. Lipid A anchors endotoxin in the outer bacterial membrane. The heteropolysaccharide, which comprises a core oligosaccharide and the O antigen, appears in the surrounding solution and determines the serological identity of the bacterium. The O antigen comprises repetitive oligosaccharide units, the composition of which is strain-specific. Characteristic building blocks of the core oligosaccharide are 2-keto-3-deoxyoctonate (KDO) and L-glycero-D-mannoheptose (Hep).
The most conservative part of endotoxin of different types is the lipid A. The inner core region is preserved similarly to lipid A, the outer core region already has a higher variation. The inner core region, KDO and lipid A itself carry a plurality of phosphate groups as substituents and are therefore responsible for the negative charge of endotoxin. Furthermore, the phosphate groups on the lipid A and on the core region can be substituted variably with arabinose, ethanolamine and phosphate. Individual saccharide building blocks of the O antigen are acetylated, sialated or glycosylated. The O antigen varies in addition with respect to the number of repetitive units, for which reason the endotoxin population of each bacterium has a certain heterogeneity.
Endotoxins are biomolecules which can be found in practically all aqueous solutions without corresponding precautionary measures. Endotoxins in humans and animals can lead to sepsis, to a strong incorrect response of the immune system. Hence, for example when producing pharmaproteins, contamination with endotoxin should be detected precisely and should be removed completely subsequently. Endotoxin represents a problem with genetically engineered pharmaceuticals, gene therapeutics or substances, which are injected into humans or animals (e.g. veterinary treatment or in animal tests). However, not only in medicinal but also in research applications, such as transfection experiments of mammal cells, inhibition or lowering of the transfection efficiency by means of endotoxin can be observed.
In order to be able to use proteins within the framework of clinical studies, the European and American pharmacopoeia demand that the proteins fall below specific boundary values for endotoxin level (e.g. immune serum globulin 0.91 EU/ml, this corresponds to 5 EU/kg bodyweight and hour (dosage=EU/kg*h); EU=endotoxin unit; FDA (Food and Drug Administration): Guideline on Validation of LAL as End Product). If a medicine or proteins contained therein have too high an endotoxin level, this can lead to the death of the experimentee. The misdirected immune defence damages the patient due to overreaction. This can lead to tissue inflammation, drop in blood pressure, heart racing, thrombosis, shock etc. Even a longer enduring endotoxin exposition in picogram quantities can lead to chronic side effects, such as e.g. immune deficiences, septic symptoms etc. Within the framework of substance production, in particular in processes with “good manufacturing practice” (GMP) conditions, it is therefore attempted to deplete endotoxin as far as possible. However, endotoxin removal in proteins, polysaccharides and DNA is problematic. In the case of proteins themselves, there are large problems due to their intrinsic properties, such as charge state or hydrophobicity, which can virtually prevent endotoxin removal or can lead to large product losses in the removal procedure.
At present, four methods for endotoxin detection in biological solutions are described, only the first two methods being permitted by the FDA. 1. “Rabbit Pyrogen Testing”; a method in which a living rabbit is injected with an endotoxin solution and hence an immune reaction is triggered. This endotoxin-induced immune response is detected by the development of fever. 2. The “Limulus Amoebocyte Lysate (LAL)”-Test, the test which is used most frequently at present (Bio Whittacker, Inc., Charles-River, Inc., Associates of Cape Cod, Inc., all USA), can be standardised in a significantly improved way. With this method, the agglomeration of the blood of the horseshoe crab (Limulus polyphemus) is measured after endotoxin contact. 3. The in vitro pyrogen test is based on the detection of interleukin-1β in human blood, which is involved in fever induction. The test consists of an incubation step of human blood with the solution to be examined, and the subsequent detection of the interleukin via antibodies. 4. A further possibility is the use of a special cell culture system (Sterogene Inc., USA) with which activation of monocytes is tracked via the appearance of specific cytokines.
The two first-mentioned methods are however very expensive and, due to the large requirement for test animals or for blood of the very rare horseshoe crab, are dubious not least on the grounds of animal protection. The LAL test can in fact also be miniaturised and automated but, due to low stability of the components, has huge disadvantages in application. Once a LAL solution has been opened it must be processed and used up immediately since the components aggregate within a few hours. The in vitro pyrogen test requires preferably fresh human blood and is relatively time consuming, as the production of the interleukin requires about 10 to 24 hours. Aside of endotoxins, other pyrogens may also be recognized with the pyrogen test. However, first of all, this test is used as substitute for the “rabbit pyrogen test”. Skilled personnel are required for all test methods and the methods are very susceptible to interference, because for example the immune system of rabbits can react entirely differently to the same dose of endotoxin. The cell culture method of the Sterogene Company, like all cell culture methods, is likewise very complex and has problems with respect to standardisation.
It can be established overall that there is no easily handled economical method for endotoxin detection and the methods used at present have a series of disadvantages. There is therefore a requirement for a method which avoids these disadvantages.